Oridonin inhibits pancreatic cancer cell migration and epithelial-mesenchymal transition by suppressing Wnt/β-catenin signaling pathway
Liu et al. Cancer Cell Int
Oridonin inhibits pancreatic cancer cell migration and epithelial-mesenchymal transition by suppressing Wnt/β-catenin signaling pathway
Qian‑Qian Liu 1
Ke Chen 1
Qiao Ye 1
Xiao‑Hua Jiang 0
YunW‑ei Sun 1
0 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong , Hong Kong SAR , China
1 Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine , Shanghai 200025 , China
Background: Oridonin (ORI) can inhibit proliferation and migration in various types of cancer cell lines. However, the exact mechanism remains unclear. We investigated the migration inhibitory effect of ORI on human pancreatic cancer SW1990 cells and dissected the possible molecular mechanism(s). Methods: CCK‑ 8 assay was used to observe the cell viability. Wound healing assay, transwell assay and spontaneous metastasis model were used to observe the migration activities. Real‑ time PCR, immunofluorescence, western blot analysis and immunohistochemistry methods were used to observe the expression of genes or proteins. Results: ORI inhibited the migration of SW1990 cells. Real‑ time PCR and immuno‑ fluorescence analyses of epithelialto‑ mesenchymal transition (EMT) markers were compared between control group and ORI group. The expression of mesenchymal molecular markers, such as vimentin, snail and slug decreased. The expression of epithelial‑ related marker E‑ cadherin increased. Wnt/β‑ catenin signalling was inhibited by ORI using luciferase reporter assay. ORI can decrease the β‑ catenin protein level not only in the nucleus, but also in the cytoplasm and the whole cell after the treatment with ORI and glycogen synthase kinase 3β (GSK3β) was increased in the ORI‑ treated group. CHIR could attenuate the effects of ORI in SW1990 cells. We established a mice model by injecting 1 × 106 SW1990 cells into nude mice intraperitoneally to test whether ORI affects tumour metastasis. Metastatic formation was inhibited by ORI (5 and 10 mg/kg) compared with the control group. Tumour sections stained with anti‑ E‑ cadherin, anti‑ vimentin and anti‑ β‑ catenin antibodies revealed that ORI inhibited EMT, as well as the Wnt/β‑ catenin pathway in vivo. Conclusions: ORI can inhibit pancreatic cancer cell SW1990 migration and EMT by down‑ regulating Wnt/β‑ catenin signal transduction in vitro and in vivo. Therefore, it can be potentially and effectively used in the clinical management of pancreatic cancer.
Oridonin; Pancreatic cancer; Metastasis; Epithelial mesenchymal transition; Wnt/β‑ catenin pathway
Pancreatic cancer, which is one of the most common
cancers of the digestive system, is a leading cause of
morbidity and mortality in both developed and
developing countries. Pancreatic cancer is characterised by a
covert anatomical structure, aggressive biology,
resistance to conventional therapeutic agents, and no early
detectable biomarkers were observed [
pancreatic cancer presents a great challenge in
oncology, with a 5-year survival rate of only 5 % [
]. This is
true for metastatic diseases, wherein the average life
expectancy is just three to 6 months. Treatment options
for pancreatic cancers are limited. Less than 20 % of
patients with pancreatic cancer are subjected to
surgical removal, whereas other patients are typically treated
with chemotherapy or chemotherapy with radiation.
Pancreatic cancer is one of the few cancers in which
survival has not improved substantially in 40 years.
Therefore, searching for new strategies to prevent and
treat pancreatic cancer is essential.
The epithelial-to-mesenchymal transition (EMT) is
among the most critical processes that occur during the
progression of tumour metastasis. EMT-induced
adenocarcinoma cells can acquire malignant features, such as
invasion, metastatic capabilities and chemo-resistance
]. In human pancreatic tumour samples, fibronectin
and vimentin are increased in high-grade tumours, with a
corresponding decrease in E-cadherin expression. These
patients have worse prognoses than those who
demonstrate less evidence of EMT. Primary tumours with
mesenchymal features (75 % of the total number of tumours)
developed metastatic lesions to the liver and lungs .
Numerous signalling pathways that are involved in the
regulation of EMT, such as transforming growth
factorbeta, notch and Wnt signalling pathways, are highly
activated in metastatic pancreatic cancers and appear to be
associated with prognosis [
Oridonin, a tetracycline diterpenoid compound
extracted from the traditional Chinese medicine
Rabdosia rubescens, has anti-inflammatory, antibacterial and
antitumour effects [
]. ORI inhibits the development of
various types of cancers and is an effective and low-toxic
antitumour medicine [
]. However, mechanisms
underlying the antitumour activities of ORI, and whether
or not it can suppress the migration of pancreatic cancer
remain largely unknown.
We investigated the effects of ORI on pancreatic
cancer cells. ORI inhibits pancreatic cancer cell migration
and EMT in vitro and in vivo by suppressing the
Wnt/βcatenin signalling pathway.
Chemicals and reagents
ORI was purchased from the Yuanye Biological
Institutions (Shanghai, China). Its purity was measured by
HPLC and determined to be 99 %. ORI was dissolved
in dimethyl sulfoxide (DMSO) to make a stock solution
(10 mM) and stored at −20 °C. The DMSO concentration
was kept below 0.1 % in all experiments and did not exert
any detectable effect on cell growth or cell death. Fetal
bovine serum (FBS), 0.25 % trypsin containing EDTA
and high glucose DMEM were obtained from Gibco
(USA). Cell Counting Kit-8 (CCK-8) was purchased from
DoJinDo (Japan). RNA extraction kit was purchased
from Life Technologies Co. CHIR99021, a GSK3β
inhibitor (R&D), was added into medium at a concentration of
2 μM 1 h before administration of ORI.
Pancreatic cancer cell lines (Aspc1, Bxpc3, Panc1,
SW1990) was obtained from the American Type
Culture Collection (ATCC; Manassas, VA, USA). The cells
were cultured in DMEM supplemented with 10 % FBS,
100U/ml penicillin, and 100 μg/ml streptomycin at 37 °C
in a humidified atmosphere of 5 % CO2, and subcultured
when confluency reached 70–80 % by 0.25 % trypsin.
The cytotoxic effect of ORI on pancreatic cancer cells
was determined by using CCK-8 kit. Briefly, the
logarithmic phase cells were plated in 96-well culture plates
(5 × 103 cells per well). After 24 h of incubation, cells
were treated with vehicle alone (0.1 % DMSO) or
various concentrations (7, 15, 31, 62, 125, 250 μM) of ORI,
followed by a 48 h-culture. Each group had 6 wells, and
CCK-8 (100 μl) was added to each well 1 h before the
end of incubation. The absorbance at 450 nm was read
using Bio-Tek, ELX800. Experiments were repeated three
times. The cytotoxic effect was expressed as a relative
percentage of cell death calculated as follows: Cell death
(%) = 100 −(dosing absorbance − blank absorbance)/
(control absorbance − blank absorbance) × 100.
Cell migration assay
A wound scratch assay was performed in order to study
the migration of SW1990 cells. A 6-well plate was seeded
with cells at 60 % confluence. After overnight
incubation, a scratch was made at the centre of each well using
a 200 μl pipette tip. Cells were further incubated with 0,
7 or 15 μM ORI. Three parallel lines were drawn at the
border of the wound, and the distance between the lines
were measured 24 h post-scratching. The microscopic
magnification (100×) of this area was photographed. The
wound healing effect was determined by measuring the
percentage of the migrating area compared with the area
of the initial wound. Each experiment was conducted in
Transwell migration assay
SW1990 cells were subjected to a transwell migration
assay. 1 × 105 cells were added to the upper chamber,
and the lower chamber was filled with 7 or 15 μM ORI of
culture medium containing no FBS. After incubation for
24 h, cells in the upper chamber were carefully removed
with a cotton swab, and cells that passed through the
membrane to the lower chamber surface were fixed and
stained with 0.5 % crystal violet. Cells from each well
were counted in 3 random high-power fields under a
microscope (100×). Each experiment was repeated in
Total cellular RNA was extracted using TRIzol
(Invitrogen) reagent. The reverse transcription was performed
with 2 μg of total RNA, using an oligo (dT) primer and
other reagents, and cDNA was synthesized using the
Revert Aid First-Strand cDNA Synthesis kit (Thermo
Fisher Scientific, Waltham, MA, USA) according to the
manufacturer’s protocol. Real-time PCR was carried out
using the miScript SYBR Green PCR kit (Qiagen, Venlo,
Netherlands). The cycle conditions for the PCR reaction
were as follows: 95 °C for 15 min followed by 40 cycles
of 60 °C for 20 s, and 72 °C for 40 s. The expression of
each RNA was normalized to that of the internal control
glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
The specific primer pairs used in this PCR reaction were:
E-cadherin (NM_004360.3), forward: 5′-ACAGCCCC
GCCTTATGATTCTC-3′, reverse: 5′-AAGCGATTGCC
CCATTCG-TT-3′; vimentin (NM_003380.3), forward:
5′-AGATGGCCCTTGACATTGAG-3′, reverse: 5′-CC
AGAGGGAGTGAATCCAGA-3′; snail (NM_005985.3),
reverse: 5′-GTCCCAGATGAGCATT-GGCA-3′; slug
(NM_003068.4), forward: 5′-GTCCGCTCTGCCGCAC
CT-3′; GAPDH (BC026907.1), forward: 5′-CTGCACC
ACCAACTGCTTAG-3′, reverse: 5′-GTCTTCTGGGTG
GCAGTGAT-3′. The changes of expression were
calculated using the (2 [−ΔΔCT]) method. Each plate was run in
After treated with or without ORI, SW1990 grown on
6-well plate were fixed in 4 % (w/v) paraformaldehyde
and permeabilized with 0.1 % Triton-X 100 in PBS.
E-cadherin (CST, 1:200), vimentin (CST, 1:200) and slug
(CST, 1:200) were visualized by sequential incubation
with indicated antibody overnight at 4 °C. Pictures were
acquired by using a fluorescence microscope (Olympus).
40 μg proteins were separated by 10 % sodium dodecyl
sulfate–polyacrylamide gel (SDS-PAGE), and blotted
onto polyvinylidene difluoride (PVDF) membranes.
The membranes were incubated with the primary
antibodies against Histone H1 (Millipore, 1:1000),
Nonphospho (Active) β-catenin (CST, 1:1000), β-catenin
(CST, 1:1000), GSK-3β(CST, 1:1000), GAPDH
(KangChen Bio-tech, 1:1000) at 4 °C overnight
separately, and further incubated with horseradish
peroxidase (HRP)-conjugated secondary antibody (1:10,000;
keyGEN-BIO) for 1 h. Bands were visualized by
Western Blotting Reagents (EMD Millipore, Billerica, MA,
Luciferase reporter assay
Sub-confluent SW1990 cells were seeded in 24-well plate
and transfected with 0.5 μg β-catenin/Tcf4 luciferase
reporter (pTop-luc) per well with Lipofectamine
(Invitrogen). After incubating for 12 h, cells were treated with
0.1 % DMSO or 15 μM ORI. 24 h later, cells were lysed
and subjected to luciferase assays using luciferase assay
kit (Promega). Each assay was done in triplicate.
Spontaneous metastasis model and Hematoxylin & Eosin (H&E) staining
SW1990 cells (1 × 106) in 50 μl PBS were injected into
the nude mice intraperitoneally. Based on the different
treatments, mice were segregated into 3 groups (each
group has 5 mice). ORI (5 mg/kg and 10 mg/kg) was
injected intraperitoneally every day for 10 days starting
from the second day of SW1990 injection. Lungs were
dissected and fixed in 10 % formaldehyde after 1 month.
Paraffin-embedded lungs were cut for H & E staining.
Immunohistochemistry analysis was performed on 5-mm
sections of formalin-fixed paraffin-embedded lungs
derived from mice. Serial sections were cut from each
tissue, and further stained to evaluate the expression of
EMT markers (E-cadherin and vimentin) and β-catenin
Quantitative results were expressed as mean ± standard
deviation (SD). The statistical analysis was performed by
using two-tailed unpaired t test (between two groups) or
one-way analysis of variance (ANOVA) (among three or
more groups) under the computer software SAS 9.2 (SAS
Institute Inc., Cary, NC, USA). A two-tailed P value <0.05
was considered to be statistically significant.
ORI inhibits cell migration in SW1990 pancreatic cell line
After treatment with different concentrations (7, 15, 31,
62, 125 or 250 μM) of ORI for 12, 24, 36 and 48 h, the
proliferation rates of four human pancreatic cancer cell
lines (Axpc1, Bxpc3, Panc1, SW1990) were assessed via
CCK-8 kit. As shown in Fig. 1a, ORI exhibited
anti-proliferative effects on all four cell lines in a
concentrationdependent manner, with SW1990 being the least sensitive
to ORI. Therefore, we decided to use this cell line, which
was derived from a spleen metastasis of a grade II
pancreatic adenocarcinoma [
], to evaluate the role of ORI
in pancreatic cancer metastasis and EMT.
IC50 of ORI in SW1990 of 24 h was 63.47 μM (Table 1).
ORI at doses higher than 30 μM inhibited cell growth.
ORI concentrations of 7 and 15 μM were used to treat
the cells for 24 h to evaluate the role of ORI in pancreatic
cancer metastasis. The effect of ORI on cell migration
was evaluated via wound healing and transwell migration
assays. As shown in Fig. 1c and e, the migrative ability of
SW1990 was significantly decreased after ORI treatment,
compared with that in the control. Treatment with the
15 μM ORI concentration resulted in 46 % reduction in
cell migrative ability (Fig. 1c; P < 0.05).
EMT of SW1990 is affected by ORI
EMT is an early event in tumour migration. It is related
to the structural changes in the intercellular junction,
which is essential for migration [
]. We hypothesised
whether the inhibition of tumour cell migration by ORI is
associated with EMT. SW1990 were treated with 15 μM
ORI for 24 h and evaluated for the expression of EMT
markers by real-time PCR and immunofluorescent
staining. Real-time PCR showed that the expression of
mesenchymal markers, such as vimentin and transcription
factors snail and slug, decreased, whereas the
expression of epithelial-related genes E-cadherin was increased
(Fig. 2a, b). However, immunofluorescent staining did
not show significant changes of E-cadherin and slug.
ORI inhibits β‑catenin signalling in human pancreatic cancer cells
Given that stabilization and nucleus translocation of
β-catenin are key events in transduction of the
canonical Wnt/β-catenin signalling, which is highly involved
in cancer metastasis, we conducted western blot
analysis to explore whether oridonin can suppress β-catenin
activity. The results indicated that ORI can decrease
the β-catenin protein level not only in the nucleus,
but also in the cytoplasm and the whole cell after the
treatment with ORI for 24 h (Fig. 3b). Luciferase assays
demonstrated that ORI dramatically suppressed the
transcriptional activity of β-catenin (Fig. 3a). The
stability of β-catenin in the cytoplasm is tightly regulated
by the Axin/APC/GSK3β complex. Phosphorylation of
β-catenin by GSK3β resulted in its degradation, which
led to the inactivation of Wnt/β-catenin signalling
].Thus, we investigated whether ORI could also
affect the expression of GSK-3β, which is the negative
regulator of Wnt/β-catenin signalling. As shown in
Fig. 3b, the total expression level of GSK3β increased
after ORI treatment. ORI may regulate Wnt/β-catenin
signalling by enhancing the function of GSK3β in
CHIR attenuates the inhibitory function of ORI
To further validate the role of β-catenin and GSK3β
in the migration inhibitory effect of ORI on SW1990
cells, SW1990 was tested with CHIR, a GSK-3-specific
inhibitor. CHIR attenuated the inhibitory function of
ORI. WB results indicated that CHIR can reverse the
decrease of the β-catenin in the cytoplasm, nucleus
and the whole cell (Fig. 4d). As shown in Fig. 4a and
b, after simulation with CHIR, the inhibited migration
of SW1990 in the ORI group (15 μM) was markedly
improved. Furthermore, the expression of E-cadherin
decreased, whereas, the expression of vimentin, snail
and slug increased after CHIR treatment (Fig. 4c, e,
P < 0.05). These results suggest that down-regulation of
β-catenin plays a critical role in the function of ORI in
ORI inhibits tumour metastasis in vivo
We injected 1 × 106 SW1990 cells into nude mice to
test whether ORI affects tumour metastasis. As shown
in Fig. 5a, ORI treatment significantly decreased the
body weight loss compared with the control. In addition,
on the fourth week, two of the five mice in the control
group died. By contrast, no death was recorded in the
ORI group (5 and 10 mg/kg). Furthermore, lung
metastasis was significantly inhibited compared with the
control group, even though no metastatic nodules could be
observed by the naked eye. A representative lung from
each group was shown in Fig. 5b. IHC analysis of tumour
sections stained with anti-E-cadherin, anti-vimentin and
anti-β-catenin antibodies revealed that ORI inhibited
EMT as well as Wnt/β-catenin pathway in vivo (Fig. 5c).
ORI is a tetracycline diterpenoid compound extracted
from the traditional Chinese medicinal plant Rabdosia
rubescens. It exerts immune protective effects in severe
inflammatory diseases. ORI has shown remarkable
antiproliferative and pro-apoptotic effects against leukemia
and some types of solid tumours [
]. It also inhibits
migration in various cancer cells [
]. ORI has been
reported to significantly inhibit lung tumour metastasis
through anti-angiogenesis by blocking Notch signalling;
it also inhibits tumour invasion and metastasis in vitro
possibly by decreasing the expression of MMPs and
regulating the Integrin β1/FAK pathway in human breast
cancer MDA-MB-231 cells. However, the effect of ORI on
the migration of pancreatic cancer cells remains unclear.
We investigated these mechanisms of ORI on pancreatic
cancer cells, and ORI can inhibit pancreatic cancer cell
SW1990 migration and EMT by down-regulating
Wnt/βcatenin signal transduction.
EMT is defined by epithelial cells taking on a
mesenchymal phenotype, characterised by loss of apical-basal
polarity, increased cellular motility and
reorganization of cytoskeleton [
]. There are three major types of
EMT: type 1 refers to embryogenesis; type 2 refers to
wound healing; and type 3 refers to cancer and
]. During the process of type 3 EMT, epithelial
cells lose contact with their neighbours and gain
mesenchymal properties, which enable them to break through
the basement membrane for their metastatic
dissemination. One of the hallmarks of EMT is the functional loss
of E-cadherin (encoded by CDH1), which is thought
to be a metastasis suppressor during tumour
]. Vimentin and the transcription factors, snail
and slug, are prominent inducers of EMT and strongly
repress E-cadherin expression [
]. Highly invasive
breast cancer cells have been studied and selected, and
these cells displayed EMT characteristics and
dramatically enhanced invasive abilities with decreased levels of
E-cadherin and increased vimentin, fibronectin, Twist
and AKT2 [
]. Metastasis may be largely dependent on
the ability of cancer cells to acquire EMT characteristics
]. Researchers found that paeoniflorin blocked the
migration and invasion of breast cancer cells by
repressing EMT under hypoxic conditions . Cui speculated
that asparaginyl endopeptidase could promote the
invasion and metastasis of gastric cancer via EMT through
AKT and MAPK signalling pathways [
]. Our results
showed that the expression of mesenchymal molecular
markers (vimentin, snail and slug) were decreased by
ORI, whereas the expression of E-cadherin increased,
indicating that ORI affects the EMT process in
pancreatic cancer cells.
Wnt/β-catenin signalling pathway plays a critical role
in various cancers [
]. In the canonical
Wnt/βcatenin signalling, Wnt ligands bind to the dual receptor
complex comprised of frizzled and low-density
lipoprotein receptor-related protein 5/6 (LRP5/6). This leads to
inactivation of the β-catenin destruction complex and
Axin/APC/GSK-3β; the critical mediator β-catenin is
relieved from its constitutive proteosomal degradation.
β-catenin subsequently accumulates in the cytoplasm
and translocates into the nucleus, where it associates
with transcription factors to regulate the downstream
target genes . Increased cytoplasmic and/or nuclear
accumulation of β-catenin protein is a common feature
in human pancreatic cancers and it is involved in its
]. The compounds from Chinese herbal
medicine targeting Wnt/β-catenin signalling can be
considered as suitable candidates for pancreatic cancer
treatment. Given that ORI could activate GSK3β
activity and up-regulate the expression of DKK1 in human
], the anti–migratory effect of ORI
on pancreatic cancer cells might result from targeting
Wnt/β–catenin signalling. Our data showed that ORI
can reduce the protein levels of active β-catenin and its
transcriptional activity in the nucleus. Moreover,
inhibiting phosphorylation of GSK3β could attenuate the
function of ORI on migration and EMT in SW1990.
In addition, ORI-treated tumour xenografts express
much lower levels of β-catenin compared with control
tumours. It is then very likely that the effect of ORI on
pancreatic cancer metastasis is due to its suppressive
effect on β-catenin signalling.
Our data suggest that ORI can be effective for human
pancreatic cancer. The anticancer effect of ORI in
SW1990 cells may result from the suppression of EMT
by increasing GSK-3β activity and inactivating
Wnt/βcatenin signal transduction. Future studies should be
directed to the identification of more ORI target proteins,
which are essential to elucidate the molecular
mechanisms underlying the anti-cancer capacity of ORI.
ORI: oridonin; EMT: epithelial‑to ‑mesenchymal transition; GSK3β: glycogen
synthase kinase 3β; DKK‑1: dickkopf‑1.
QQL was the main experimental investigator and had drafted the manuscript.
QY participated the data analysis. KC helped to complete the molecular
experiments. XHJ and YWS supervised the study and the manuscript. All
authors read and approved the final manuscript.
We thank Professor Tingjun Ye for his excellent academic assistance.
The authors declare that they have no competing interests.
The authors received no financial support for the research, authorship, and/or
publication of this article.
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